Erythropoietin (EPO) is a polypeptide rich in sugar chains, which is predominantly produced in the kidneys and acts on precursor cells of hematopoietic tissue to stimulate their differentiation and proliferation into erythrocytes. EPO is currently commercially available as human EPO recombinantly produced in animal host cells, and its main use is as a therapeutic agent for various types of anaemia, including renal anemia caused by the underproduction of EPO resulting from nephropathy.
As used herein, the term “native EPO” is intended to encompass human urine-derived EPO, such as those extracted, isolated and purified by various techniques, and recombinant human EPO (rhEPO) having the same sugar chains as human-derived EPO, such as those recombinantly produced in animal host cells (e.g., CHO cells, COS cells), as well as their variants modified to include substitution or deletion of one or more amino acids constituting such EPOs or modified to include addition of one or more amino acids.
Currently used EPO is administered, e.g., by intravenous, subcutaneous or intramuscular route. The percentage of reticulocytes (erythrocyte precursors) in total erythrocytes can be used as an indicator for EPO activity. The activity of native EPO observed as the percentage of reticulocytes will reach a peak 3 to 5 days after administration by any route mentioned above, followed by a rapid decline. Thus, native EPO should be injected twice or three times per week to ensure sufficient therapeutic effects in anaemia patients. This not only causes pain in the patients, but also provides additional load on overworked doctors and other medical staff members. Further, a decreased number of injections required within a fixed period of time will save medical costs.
On the other hand, there are reports on proteins or glycoproteins conjugated with water-soluble polymers (e.g., PEG) having a hydrazide or oxylamine moiety capable of covalent bonding through chemical reaction with an oxidizable functional group such as polyol, lactol, amine, carboxylic acid or a carboxylic acid derivative on the proteins or glycoproteins (see, e.g., JP 7-196925 A). According to this report, a water-soluble polymer, such as PEG, can be attached to various free radicals on amino acids or sugar residues constituting a protein or glycoprotein to yield a coupling product with 6 to 34 PEG molecules (molecular weight: 2000 to 12000) per protein molecule. In such a PEG-conjugated protein or glycoprotein with many PEG molecules, it is difficult to control the positions and number of PEG molecules attached to the protein or glycoprotein and it is hard to obtain an uniform PEG-conjugated protein or glycoprotein. Thus, there is a problem when the PEG-conjugated protein or glycoprotein thus prepared is formulated into pharmaceuticals.
There is also a report disclosing a sulfonate ester-activated polymer (e.g., sulfonate ester-activated PEG) and proposing a method in which this sulfonate ester-activated polymer is reacted with a target material (e.g., a protein or glycoprotein) to prepare a polymer-conjugated target material (see JP 9-504515 A). Examples of a reactive group on a target material which reacts with this sulfonate ester-activated polymer include a primary or secondary amino group, a thiol group and an aromatic hydroxyl group. In such a sulfonate ester-activated polymer capable of reacting with various reactive groups, it is therefore regarded as difficult to control the number and positions of conjugatable polymer molecules which are introduced. Also, such a polymer clearly has the possibility of sulfonate amide formation, which allows much higher heterogeneity for products.
Further, there is a report on a branched polymer (e.g., branched PEG) which is attached to a target material (e.g., a protein or glycoprotein) to give a branched polymer conjugate of the target material (see JP 9-504299 A). Although this branched polymer-conjugated target material also successfully sustains its efficacy, it has been desired to develop polymer-conjugated EPOs with more sustained efficacy.
Also, it has been believed that conjugation with higher molecular weight PEG would result in more sustained efficacy.
Conjugation with higher molecular weight PEG will cause a larger decrease in the in vitro activity of EPO. However, it has been believed that EPO conjugated with higher molecular weight PEG would show significantly improved plasma retention and hence sustained in vivo activity, resulting in greater and more sustained activity with increase in the molecular weight of PEG (Polyethylene glycol-conjugated pharmaceutical proteins; PSTT Vol. 1, No. 8, 1998, 352-356). For example, in the case of a PEG conjugate of G-CSF mutaine, it is known that its in vivo activity increases in proportion to the calculated molecular weight of PEG in the range of about 10 kDa up to 70 kDa (PCT/US00/01264, WO 00/44785).
Conventional PEG conjugates of EPO were designed to have many PEG molecules with a relatively low molecular weight of around 5 kDa in order to sustain their efficacy. However, in EPO rich in sugar chains, conjugation was limited exclusively to unglycosylated amino acid residues involved in receptor binding, making it impossible to avoid a decrease in in vivo activity and difficult to balance sustained efficacy and decreased in vivo activity. Also, even in a case where amino groups or sugar chains are conjugated with PEG of greater than 10 kDa as stated above, there has been a difficulty in practically formulating PEG-conjugated EPO into drugs because the problem of controlling the number of PEG molecules still remains.